1. Field of the Invention
The present invention relates to a diaphragm and a device for measuring cellular potential, which is used for measuring an electrophysiological activity of cells, and a manufacturing method of the diaphragm.
2. Background Art
A patch clamp technique is one of conventional methods for elucidating a function of an ion channel existing in a cell membrane or screening (examining) medicines with electrical activities of cells as a reference mark. In the patch clamp technique, a small portion (a patch) of the cell membrane is slightly sucked by a tip portion of a micropipette. Then, by using a fine electrode probe provided in the micropipette, electric current flowing across the patch in the fixed membrane potential is measured. Thus, opening and closing state of one or a few ion channels existing in the patch is electrically measured. This method is one of a few-number of methods capable of investigating a physiological function of a cell on real time basis.
However, the patch clamp technique requires a special technique and skill for preparation and operation of the micropipette, and much time is required to measure one sample. Therefore, this technique is not suitable for an application that requires high-speed screening of a large amount of candidate compounds for a medicine. On the other hand, recently, a flat-shaped fine electrode probe using a fine processing technology has been developed. Such a fine electrode probe is suitable for an automated system that does not require insertion of a micropipette for each individual cell. Hereinafter, the example thereof is described.
For example, Japanese Translation of PCT Publication NO. 2002-518678 discloses a technology for measuring potential-dependent ion channel activities of a test cell attached to an opening of a through-hole by an electrode disposed on the lower side of a plurality of through-holes provided in a cell holding substrate. Furthermore, recently, there has been disclosed a technology for measuring extracellular potential with high degree of accuracy by forming a through-hole of 2.5 μm inside a cell holding substrate made of silicon oxide and allowing this through-hole to hold HEK293 cell which is a kind of human cultured cell line, so as to secure high adhesiveness.
Published PCT International Applications No. 02/055653 pamphlet discloses device 1 for measuring cellular potential shown in FIG. 29. Device 1 for measuring cellular potential includes substrate 2 and well 3 disposed on the upper side of substrate 2. On the upper surface of substrate 2, depression 4 is formed. Through-hole 5 penetrating from the lower part of depression 4 to the lower surface of substrate 2 is provided. In well 3, first electrode 6 is disposed. In through-hole 5, second electrode 7 is disposed. Furthermore, second electrode 7 is connected to a signal detector via wiring 8.
Next, an operating method of device 1 for measuring cellular potential is described. Firstly, test cell (hereinafter, referred to as “cell”) 10 and electrolyte 9 are filled in well 3. Cell 10 is captured and held by depression 4. When measurement is carried out, cell 10 is sucked with a suction pump or the like from the lower side of through-hole 5 and held in a state in close contact with an opening of through-hole 5. That is to say, through-hole 5 plays the same role as a tip hole of a glass pipette. The function, pharmacological reaction, or the like of the ion channel of cell 10 can be analyzed by measuring voltage or current between first electrode 6 and second electrode 7 before and after the reaction so as to calculate the potential difference between the inside and outside of cell 10. As mentioned above, by providing depression 4, even when thick substrate 2 is used for securing mechanical strength, the length of through-hole 5 is reduced, and the processing becomes easier. Furthermore, suction force to cell 10 from the lower side of substrate 2 is increased.
However, it has been not possible to control a position of through-hole 5 with high degree of accuracy, conventionally. Consequently, it is not possible to control depth of depression 4 and through-hole 5 with high degree of accuracy. As a result, length of through-hole 5 tends to vary, so that it may be impossible to bring cell 10 into close contact with through-hole 5 appropriately. When cell 10 is sucked, pressure applied to cell 10 becomes short depending upon the length of through-hole 5. As a result, cell 10 may be damaged or the adhesiveness (seal resistance) between cell 10 and through-hole 5 may be reduced. Thus, the measurement accuracy of device 1 for measuring cellular potential may be reduced.